Heregulin-alpha (HRGalpha) is a cytokine secreted by the mammary mesenchyme, adjacent to lobuloalveolar structures. To understand the role of HRGalpha and its receptors in mammary glands, and the underlying mechanisms, we performed this study to determine the expression and localization of HRGalpha and its receptors ErbB2 and ErbB3. We also determined the role of HRGalpha in the development of mammary glands, beta-casein expression and secretion, Rab3A protein expression and the phosphorylation of HRGalpha signaling molecules using confocal laser scanning microscopy, tissue culture, capillary electrophoresis, Western blotting and enzyme-linked immunosorbent assays. We found that a peak was on pregnancy day 15. Changes of ErbB2 and ErbB3 expression were positively and linearly correlated with HRGalpha, indicating that HRGalpha positively regulates ErbB2 and ErbB3 expression. During pregnancy, HRGalpha enhanced the phosphorylation of STAT5, p42/p44, p38, PKC and Rab3A protein expression, stimulated the proliferation and differentiation of the ductal epithelial cells of mammary glands, and increased and maintained the expression and secretion of beta-casein. During lactation, HRGalpha enhanced the phosphorylation of STAT5 and p38, inhibited the phosphorylation of PKC and Rab3A protein expression, maintained the morphology of the mammary glands and increased the secretion of lactoprotein to reduce the expression of beta-casein in mammary epithelial cells. During involution, HRGalpha induced the phosphorylation of STAT3 and Rab3A protein expression, and inhibited the phosphorylation of PKC to stimulate the degeneration of mammary epithelial cells. It also inhibited the secretion of beta-casein, resulting in increased levels of beta-casein in mammary epithelial cells.